1. Field of the Invention
The present invention relates to methods and nucleic acid compositions for selectively inhibiting gene expressing, involving the preparation and use of anti-sense RNA molecules that encode sequences complementary to distinct intron regions for the inhibition of, for example, oncogene expression.
2. Description of the Related Art
It is now well established that a variety of diseases, ranging from certain cancers to various genetic defects, are caused, at least in part, by genetic abnormalities that result in either the over expression of one or more genes, or the expression of an abnormal or mutant gene or genes. For example, many forms of cancer in man are now known to be the result of, at least indirectly, the expression of xe2x80x9concogenesxe2x80x9d. Oncogenes are genetically altered genes whose altered expression product somehow disrupts normal cellular function or control (Spandidos, et al., 1989).
Most oncogenes studied to date have been found to be xe2x80x9cactivatedxe2x80x9d as the result of a mutation, often a point mutation, in the coding region of a normal cellular gene or of a xe2x80x9cprotooncogenexe2x80x9d, that results in amino acid substitutions in the protein expression product. This altered expression product, in turn, exhibits an abnormal biological function that somehow takes part in the neoplastic process (Travali, et al., 1990). The underlying mutations can arise by various means, such as by chemical mutagenesis or ionizing radiation.
A number of oncogenes have now been identified and characterized to varying degrees, including ras, myc, neu, raf, erb, src, fms, jun and abl (Travali, et al., 1990; Minna, 1989; Bishop, 1987). It is quite likely that as our knowledge of tumorigenesis increases, additional oncogenes will be identified and characterized. Many of the foregoing, including ras, myc and erbB, comprise families of genes, whose expression product bear sequence similarities to other members of the family (Shih, et al., 1984; Bos, 1989; Schwab, et al., 1985). In the case of many of these gene families, it is typical that oncogenesis involves an activation of only one member of the family, with other xe2x80x9cunactivatedxe2x80x9d members serving a role in normal cellular functions
The study of DNA-mediated gene transfer has revealed the existence of activated cellular oncogenes in a variety of human tumors (for review, see Cooper, et al., 1982). Oncogenes have been identified in human bladder, colon, lung and mammary carcinoma cell lines (Krontiris, et al., 1981; Murray, et al., 1981; Perucho, et al., 1981), promyelocytic leukemia (Murray, et al., 1981), neuroblastoma (Shimizu, et al., 1983) and sarcoma cell lines (Pulciani, et al., 1982), and various solid tumors including carcinomas of the lung, and pancreas (Pulciani, et al., 1982). Studies have demonstrated that various transforming genes detected by transfection correspond to activated cellular homologues of retroviral oncogenes (Pulciani, et al., 1982; Der, et al., 1982; Parada, et al., 1982; Santos, et al., 1982), although others have no known retroviral cognate (Tulciani, et al., 1982; Lane, et al., 1982).
The ras oncogene family has been perhaps the best characterized to date (Barbacid, 1987; Bos, 1989). Most of the identified transforming genes in human carcinomas have been a member of the ras gene family, which encode immunologically related proteins having a molecular weight of 21,000 (p21) (Ellis, et al., 1981; Papageorge, et al., 1982). This family is comprised of at least 3 members, one transduces as H-ras in the Harvey strain of murine sarcoma virus (Ellis, et al., 1981), one as K-ras and Kirsten murine sarcoma virus (Ellis, et al., 1981), and one identified by low stringency hybridization to H-ras, termed N-ras (Shimizu, et al., 1983). As noted, all members of the ras gene family encode closely related proteins of approximately 21,000 Daltons which have been designated p21s (Ellis, et al, 1981). The level of p21 expression is similar in many different human tumor cells, independent of whether the cell contains an activated ras gene detectable by transfection.
Nucleotide sequence analysis of the H-ras transforming gene of the EJ human bladder carcinoma has indicated that the transforming activity of this gene is a consequence of a point mutation altering amino acid 12 of p21 from glycine to valine (Tabin, et al., 1982). Studies of proteins encoded by K-ras genes activated in four human lung and colon carcinoma cell lines indicated that the transforming activity of K-ras in these human tumors was also a consequence of structural mutations (Der and Cooper, 1983). Other mutations have been found to result in ras gene activation as well. For example, the H-ras gene activated in a lung carcinoma cell line encodes the normal amino acid position 12 but is mutated at codon 61 to encode leucine rather than glutamine (Yuasa, et al., 1983). An N-ras gene activated in a human neural blastoma cell line is also mutated at codon 61 but encodes lysine rather that glutamine (Taparowski, et al., 1983). Thus, studies such as these have indicated that ras genes in human neoplasms are commonly activated by structural mutations, often point mutations, that thus far occur at codon 12 or 61 with different amino acid substitutions resulting in ras gene activation in different tumors.
Antisense RNA technology has been developed as an approach to inhibiting gene expression, particularly oncogene expression. An xe2x80x9cantisensexe2x80x9d RNA molecule is one which contains the complement of, and can therefore hybridize with, protein-encoding RNAs of the cell. It is believed that the hybridization of antisense RNA to its cellular RNA complement can prevent expression of the cellular RNA, perhaps by limiting its translatability. While various studies have involved the processing of RNA or direct introduction of antisense RNA oligonucleotides to cells for the inhibition of gene expression (Brown, et al., 1989; Wickstrom, et al., 1988; Smith, et al., 1986; Buvoli, et al., 1987), the more common means of cellular introduction of antisense RNAs has been through the construction of recombinant vectors which will express antisense RNA once the vector is introduced into the cell.
A principle application of antisense RNA technology has been in connection with attempts to affect the expression of specific genes. For example, Delauney, et al. have reported the use antisense transcripts to inhibit gene expression in transgenic plants (Delauney, et al., 1988). These authors report the down-regulation of chloramphenicol acetyl transferase activity in tobacco plants transformed with CAT sequences through the application of antisense technology.
Antisense technology has also been applied in attempts to inhibit the expression of various oncogenes. For example, Kasid, et al., 1989, report the preparation of recombinant vector construct employing Craf-1 cDNA fragments in an antisense orientation, brought under the control of an adenovirus 2 late promoter. These authors report that the introduction of this recombinant construct into a human squamous carcinoma resulted in a greatly reduced tumorigenic potential relative to cells transfected with control sense transfectants. Similarly, Prochownik, et al., 1988, have reported the use of Cmyc antisense constructs to accelerate differentiation and inhibit G1 progression in Friend Murine Erythroleukemia cells. In contrast, Khokha, et al., 1989, discloses the use of antisense RNAs to confer oncogenicity on 3T3 cells, through the use of anitisense RNA to reduce murine tissue inhibitor or metallo proteinases levels.
Unfortunately, the use of current antisense technology often results in failure, particularly where one seeks to selectively inhibit a member of a gene family. This is presumably due to the similarity in underlying DNA sequence, which results in the cross-hybridization of antisense RNA, which retards the expression of genes required for normal cellular functions. An example is presented by Debus, et al., 1990, who reported that in the case of ras oncogenes, antisense ras oligonucleotides kill both normal and cancer cells, which, of course, is not a desired effect.
Therefore, while it is clear that antisense technology shows potential promise as a means of external control of gene expression, it is equally clear that it does suffer particular draw backs, such as in its lack of selectivity where gene families are concerned. There is a particular need, therefore, for a general approach to the design of antisense RNA which will allow selective inhibition of gene expression, even in the case of closely related genes.
The present invention, in a general and overall sense, addresses one or more of the foregoing or other shortcomings in the prior art by providing a novel approach to the design of antisense RNA molecules, and their coding sequences, in a manner which allows their use to inhibit the expression of specific genes. The inventors believe that the approach offered by the present invention offers more specificity and selectivity than previous approaches. Additionally, it is proposed that the present invention will allow that the development of antisense technology having a much improved ability to inhibit specific gene expression, particularly in those instances where one desires to selectively inhibit a particular gene over that of closely related genes or other members of a gene family.
A principle feature of the present invention is the antisense RNA molecules themselves, which include a region that is complementary to and is capable of hybridizing with an intron region of the gene whose expression is to be inhibited. The inclusion of intron-complementary regions in the antisense RNA constructs of the present invention is believed to be the key to both an improved inhibitory capability as well as selectivity. By way of theory, it is proposed that the use of antisense intron regions provides an improved capability for at least two reasons. It is known that the structure of intron RNA plays a role in RNA processing.
The inventors propose that antisense introns bind to xe2x80x9csensexe2x80x9d intron regions found on the initial RNA transcript of the gene, an affects proper RNA processing. Thus, subsequent translation of protein-coding RNAs into their corresponding proteins is retarded or prevented. The use of antisense introns are believed to provide selectivity of inhibition because the exon or xe2x80x9camino acid encodingxe2x80x9d region of RNAs coding for closely related proteins are often themselves closely related. This may not be the case for the introns of closely related genes. Thus, where intron regions between two genes are distinct, antisense introns can be designed which will hybridize selectively to a selected gene family member, and not to other family members, and thereby inhibit selectivity.
As used herein, the term xe2x80x9cintronxe2x80x9d is intended to refer to gene regions that are transcribed into RNA molecules, but processed out of the RNA before the RNA is translated into a protein. In contrast, xe2x80x9cexonxe2x80x9d regions of genes are those regions which are transcribed into RNA and subsequently translated into proteins.
Thus, where one seeks to selectively inhibit a particular gene or genes over a related gene or genes, the inventors propose the preparation and use of antisense RNA molecules which encode an intron region or regions of the gene which one desires to inhibit selectively, that is distinct from intron regions of genes which one desires to leave unaffected. A xe2x80x9cdistinctxe2x80x9d intron region, as used herein, is intended to refer to an intron region that is sufficiently different from an intron region of another gene such that no cross hybridization would occur under physiologic conditions. Typically, where one intron exhibits a sequence homology of no more than 20% with respect to a second intron, one would not expect hybridization to occur between antisense and sense introns under physiologic conditions.
While it is generally preferred that antisense introns be prepared to be complementary to an entire intron of the gene to be inhibited, it is believed that shorter regions of complementarity can be employed, so long as the antisense construct can be shown in vitro to inhibit expression of the targeted expression product. The inventors believe that the most important intron regions in terms of the preparation of antisense introns will be those regions closest to intron/exon junctions. This is the region where RNA processing takes place. Thus, it is proposed that one will desire to include it in the antisense intron sufficient complementarity with regions within 50-100 nucleotides of the intron/exon junction.
The inventors have found that some antisense exon sequences of the targeted gene can also be included in the antisense. constructs of the present invention, so long as the resultant construct maintains its selectivity, and will not seriously inhibit genes whose continued function is relied upon by the cell for normal cellular activities. The amount of antisense exon sequence included within the antisense construct which can be tolerated will likely vary, depending on the particular application envisioned. For example, antisense constructs for down-regulation of K-ras expression have been prepared which include sequences complementary to exons II and III and all of intron II of the K-ras gene. These constructs contain antisense sequences to intron II of K-ras, and selectively inhibit K-ras expression relative to H-ras or N-ras. Thus, in this instance, the inclusion of sequences complementary to exons II and III of K-ras apparently did not result in the significant inhibition of the H-ras or N-ras genes, even though a 300 nucleotide region of complementarity existed with exons of the unaffected genes.
One can readily test whether too much antisense exon DNA has been included in antisense intron constructs of the present invention by simply testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences are affected.
It is proposed that the antisense constructs of the present invention, whether they be the antisense RNA molecules (i.e., olligonucleotides) or nucleic acid molecules which encode for antisense RNA molecules, will have their principal application in connection with the down-regulation of oncogene expression. The most preferred oncogenes for application of the present invention will be those which exist as a family of genes, where one desires to selectively inhibit one member of a family over other members. In this regard, one may mention by way of example, the ras, myc, erb or jun families of oncogenes. Certain of these, such as the ras family, involves the activation of protooncogenes by a point mutation, which apparently results in the expression of a biologically abnormal product.
The present invention contemplates that antisense intron RNA can either be applied directly to cells, in the form of oligonucleotides incorporating antisense intron sequences, or by introducing into the cell nucleic acid sequences that will encode the desired antisense construct. In the former case, it has been shown by others that antisense oligonucleotides can successfully traverse cellular membranes. The present inventors envision that such an approach may be an option to therapy, particularly where the antisense oligonucledtides are successfully packaged to maintain their stability in circulation, for example, by liposome encapsulation.
Other techniques for direct insertion in the cells include, by way of example, electroporation, or calcium phosphate transfection. Furthermore, where one desires to treat conditions of the bone marrow, bone marrow cells can be successfully removed from the body, treated with antisense constructs, and replaced into the body similar to the adoptive immunotherapy approach employed in IL-2 treatment.
It is proposed that a more preferred approach will involve the preparation of vectors which incorporate nucleic acid sequences capable of encoding the desired antisense intron construct, once introduced into the cells to be treated, preferably, these sequences are stably integrated into the genome of the cell. One example of such of vector construct would be a replication defective retrovirus, such as LNSX, LN or N2A, that is made infective by appropriate packaging, such as by GPtenvAM-12 cells. Although the retrovirus would inhibit the growth of the tumor, the expression of the antisense construct in non-tumor cells would be essentially harmless where one prepares a retrovirus construct which encode distinct antisense intron RNA in accordance with the present invention. In addition to retroviruses, it is contemplated that other vectors can be employed, including adenovirus, adeno-associated virus, or vaccinia viruses (Hermonat, et al., 1984; Karlsson, et al., 1985; Mason, et al., 1990).
Therefore, in certain aspects, the present invention contemplates the preparation of nucleic acid molecules which comprise a coding region capable of expressing an antisense xe2x80x9cintronxe2x80x9d RNA molecule having regions complementary to and capable of hybridizing with an intron region of selected gene. Generally speaking, preferred nucleic acid molecules will be DNA sequences arranged in a vector, such as a virus or plasmid, and positioned under the control of an appropriate promoter. However, antisense RNA can be itself an RNA molecule, such as retrovirus RNA into which the appropriate coding sequences have been incorporated. In either case, the nucleic acid encoding sequences will be arranged in a vector that will preferably be capable of stably integrating the antisense coding sequences into the genome of the targeted cell.
The particular promoterthat is employed to control the expression of the antisense RNA in a vector construct is not believed to be particularly crucial, so long as it is capable of expressing the antisense intron RNA in the targeted cell of a rate greater than 5 fold that of the gene to be inhibited. Thus, where a human cell is targeted, it will be preferred to position the antisense RNA coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human cellular or viral promoter. By way of example, one may mention the RSV, N2A, LN, LNSX, LNSN, SV40, LNCX or xcex2-actin promoter (Miller, et al., 1989; Hamtzoponlos, et al., 1989).
The most preferred promoters will be those that are capable of being expressed in a wide variety of histologic cell types, and which is capable of continuously expressing the antisense RNA. A preferred example is the xcex2-actin promoter, because the promoter functions effectively in human epithelial cells. Other examples of promoters having a similar capability include RSV and SV40.
In further aspects, the present invention concerns a method of selectively inhibiting the expression of a gene product of a selected gene in a cell, which includes preparing an antisense RNA molecule having a region that is complementary to and capable of hybridizing with a distinct intron region of the selected gene, followed by introducing the antisense RNA into the cell in an amount effective to inhibit the expression of the gene product.
In still further embodiments, the invention concerns a method for the inhibition of tumorigenicity of ras-transformed cells, which includes a first step of testing the cell to identify the particular ras gene that has been activated, preparing an antisense RNA molecule which includes a distinct intron of the activated ras oncogene that is not found in an intron of ras genes which are not activated in the cell, and introducing the antisense RNA into the cell in amounts effective to selectively inhibit the activated ras gene. The inventors have found that the invention has particular applicability to control of ras gene expression, particularly K-ras, and have shown that the expression of a particular ras gene can be effectively inhibited, without affecting cell viability.
In still further embodiments, the present invention relates to methods of preparing genetic constructs for the expressing of antisense intron DNA, which includes incorporation of genomic DNA fragments, as opposed to cDNA, into appropriate vectors for subsequent intracellular incorporation. Of course, the use of cDNAs alone in the preparation of antisense RNA will not be in accordance with the present invention, in that, by definition, cDNAs will not include the required intron sequences. However, intron sequences will be represented in genomic DNA, which therefore provides a useful source of DNA fragments for application to the present invention.